In vivo targeting of dendritic cells

ABSTRACT

The invention provides a composition for modulating immunity by the in vivo targeting of an antigen to dendritic cells. The composition comprises: a preparation of antigen-containing membrane vesicles or antigen-containing liposomes which have on their surfaces a plurality of metal chelating groups; and, a ligand for a receptor on the dendritic cells, the ligand being linked to a metal chelating group via a metal affinity tag on the ligand. The composition further includes an immunomodulatory factor. A process for preparing the composition is also provided. The invention further provides a method of modulating an immune disorder, and methods of treating tumours and infections.

TECHNICAL FIELD

The invention described herein relates generally to the composition ofpreparations for the targeting of membrane-associated antigen (Ag) todendritic cells (DCs) in order to modulate immune responses, either fordisease prevention or for therapeutic purposes. More particularly, theinvention relates to a method of modifying Ag-containing membranes, toenable engraftment and/or incorporation of targeting molecules andimmunomodulatory factors, allowing the modified membranes to be targetedto DCs in vivo and potently induce, or suppress, immune responses. Evenmore particularly, the invention relates to a composition that can beused to modify Ag-containing membrane structures, such as liposomes orplasma membrane vesicles (PMVs), to enable the membranes to be targetedeffectively to DCs in vivo, thereby modulating immunity, and enablingthem to be used either as vaccines or vaccine-like agents inimmunotherapies to prevent or treat disease in humans and animals.

BACKGROUND ART

Dendritic cells (DCs) are a rare population of antigen presenting cells(APCs) uniquely capable of stimulating primary immune responses, and astrong interest has developed in their use in cancer immunotherapies.¹Attempts to harness the capacity of DCs to stimulate potent immuneresponses have hitherto focused primarily on procedures involving themanipulation of DCs ex vivo. This approach often requires that DCs beisolated from a patient, expanded in numbers, loaded with antigen (Ag)(ref's 2-5), and then be re-introduced into the patient. While thisprocedure is simple in principle, there are difficulties associated withisolation and culture of such a rare cell population.^(6,7) Clearly,strategies that deliver Ags directly to DCs in vivo, and that can elicitan appropriate immune response, have enormous clinical potential.

DCs originate from progenitors in the bone marrow and migrate asimmature cells to peripheral tissues where they internalise Ag andundergo a complex maturation process. Ag is internalised via a number ofsurface receptors, including the complement receptors (e.g., CD11c/CD18)and the endocytic receptors (e.g., DEC-205, DC-SIGN and Toll-likereceptors). During Ag acquisition, immature DCs also may receive “dangersignals”, in the form of pathogen-related molecules such as bacterialcell wall lipopolysaccharide (LPS), or inflammatory stimuli viacytokines such as IFN-γ. DCs then migrate to the secondary lymphoidorgans, maturing to become competent APCs.⁸ Receptors such asCD11c/CD18, DEC-205, DC-SIGN and Toll-like receptors play a crucial rolein the process of Ag capture and presentation, and are expressedprimarily on DCs. It is conceivable, therefore, that these receptorsalso could be used for targeting Ag directly to DC in vivo. Consistentwith this notion, fusion proteins composed of Ag and single chainantibodies (ScFvs) to DEC-205 have been shown to target to DCs in vivo,inducing T cell activation when co-administered with inflammatorystimulators such as anti-CD40 antibody.^(9,10) In contrast, in theabsence of such inflammatory stimulators, antigen targeted to DCs viathe ScFv induced T cell unresponsiveness.

Synthetic liposomes have the potential to deliver large quantities ofAgs to DCs (Ref. 11), but to date their targeting to specific DC surfacemolecules has been difficult to achieve in practice.^(12,13) Clearly, aneffective strategy that combines the Ag carrying capacity of liposomesand the specificity of molecular recognition to target multiple Agseither with or without “danger signals” directly to DCs in vivo, wouldhave enormous potential in simplifying DC immunotherapies, particularlyfor cancer, infections, and autoimmune diseases.

In International Application No. PCT/AU00/00397 (Publication No. WO00/64471) there is described a method of modifying biological orsynthetic membranes or liposomes for the purposes of altering immunity,or for the targeting of drugs and other agents to a specific cell typeor tissue when the modified biological or synthetic membranes orliposomes are administered in vivo. Modification of the membranes orliposomes is achieved by the incorporation or attachment of metalchelating groups, thereby allowing engraftment of one or more targetingmolecules possessing a metal affinity tag. However, the nature of theimmune response induced by targeting Ag to DCs is critically dependenton the presence of specific immunomodulatory factors such as cytokinesor “danger” signals, and there is no disclosure or suggestion inPCT/AU00/00397 of the membrane modification that is required, or theimmunomodulatory factors that are needed, to elicit an appropriateimmune response in vivo.

SUMMARY OF THE INVENTION

An object of the invention the subject of this application, is toprovide a composition for the in vivo targeting to DCs, of Ag-containingliposomes and PMV, by modifying the said membranes through incorporationof an appropriate immunomodulatory factor, or “danger signal”, and theengraftment of a ligand, that can target the modified membranes toreceptors on the surface of DCs, and hence elicit an appropriate immuneresponse. The composition can be used as vaccines or in immunotherapies,either to potentiate immunity for preventing or treating diseases suchas various cancers and infections, or to suppress immunity to a specificself Ag in a way that can be used to treat or prevent transplantrejection, or the effects of autoimmune diseases such as type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus andmultiple sclerosis.

Further objects of the invention are to provide a process for preparingsuitable compositions, and methods of treatment utilising thecompositions.

According to a first embodiment of the invention, there is provided acomposition for modulating immunity by the in vivo targeting of anantigen to dendritic cells, the composition comprising:

a preparation of antigen-containing membrane vesicles orantigen-containing liposomes having on the surface thereof a pluralityof metal chelating groups; and

a ligand for a receptor on said dendritic cells, said ligand beinglinked to a said metal chelating group via a metal affinity tag on saidligand; wherein,

said antigen-containing vesicles or liposomes include animmunomodulatory factor.

According to a second embodiment of the invention, there is provided aprocess for preparing a composition for modulating an immune response bythe in vivo targeting of an antigen to dendritic cells, the processcomprising the steps of:

-   i) preparing antigen-containing membrane vesicles or    antigen-containing liposomes;-   ii) modifying said antigen-containing membrane vesicles or    antigen-containing liposomes by the incorporation of at least one    immunomodulatory factor;-   iii) further modifying said antigen-containing membrane vesicles or    antigen-containing liposomes by the incorporation of amphiphilic    molecules, wherein said amphiphilic molecules include a chelator    group which lies on the surface of said antigen-containing membrane    vesicles or antigen-containing liposomes when incorporated therein;    and-   iv) contacting the product of step (iii) with a ligand for a    receptor on said dendritic cells, wherein said ligand includes a    metal affinity tag for binding to said chelator group.

According to a third embodiment of the invention, there is provided amethod of modulating an immune response in a subject, the methodcomprising administering to said subject a composition according to thefirst embodiment.

According to a fourth embodiment of the invention, there is provided amethod of preventing or treating a tumour in a subject, the methodcomprising administering to the subject a composition according to thefirst embodiment, wherein said antigen included in saidantigen-containing membrane vesicles or antigen-containing liposomes isa tumour antigen.

According to a fifth embodiment of the invention, there is provided amethod of preventing or treating an infection in a subject, the methodcomprising administering to the subject a composition according to thefirst embodiment, wherein said antigen included in saidantigen-containing membrane vesicles or antigen-containing liposomes isan antigen from an agent causing the infection.

According to a sixth embodiment of the invention, there is provided useof a composition according to the first embodiment in the preparation ofa medicament for modulating an immune response in a subject.

According to a seventh embodiment of the invention, there is provideduse of a composition according to the first embodiment in thepreparation of a medicament for preventing or treating a tumour in asubject.

According to an eighth embodiment of the invention, there is provideduse of a composition according to the first embodiment in thepreparation of a medicament for preventing or treating an infection in asubject.

Other embodiments of the invention will become apparent from a readingof the following detailed description of the invention, in whichdescription there will be reference to the accompanying drawings brieflydescribed hereafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the novel chelator lipid (NTA)₃-DTDA. FIG.1B is a schematic representation of the (NTA)₃-DTDA lipid incorporatedin antigen (Ag) containing stealth liposomes (SL) composed ofpalmitoyl-oleoyl-phosphatidylcholine (POPC) andphosphatidyl-ethanolamine-(polyethylene) glycol₂₀₀₀ (PE-PEG₂₀₀₀). FIG.1C is similarly a schematic representation of liposomes of similarcomposition to those of FIG. 1B but without PE-PEG₂₀₀₀ can be fused withantigen-bearing tumour cell-derived plasma membrane vesicles (PMV). Inboth instances, SL (B) and modified PMV (C), the lipid tracer PC-BODIPY(not shown) can also be included to facilitate tracking of either theliposomes or the modified PMV. The (NTA)₃-DTDA permits the engraftmentof histidine-tagged ScFv Abs against DEC-205 and CD11c onto the liposomeor modified PMV surface, and consequently, the targeting of these tosurface markers such as DEC-205 and CD11c on DCs.

FIG. 2 shows that PMV and SL engrafted with CD11c-ScFv and DEC-205-ScFvbind to DCs. With regard to FIG. 2A, PMV derived from B16-OVA cells werefused with liposomes composed of POPC, (NTA)₃-DTDA, and PC-BODIPY. ThePMV were then engrafted with a control peptide (PMV-L2), CD11c-ScFv(PMV-CD11c), or DEC-205-ScFv (PMV-DEC-205), before being incubated withLTC-DC and cell bound BODIPY-fluorescence quantified by flow cytometry.FIG. 2B shows the binding to LTC-DC of similarly-engrafted SL composedof POPC, (NTA)₃-DTDA, PE-PEG₂₀₀₀ and PC-BODIPY. Each profile isrepresentative of that obtained from three separate experiments.

FIG. 3 shows that ScFv-engrafted PMV target DCs in draining lymph node.Mice were injected in the hind footpad with fluorescein-labelled PMVthat had been engrafted with either control protein (PMV-L2), or withScFv to CD11c (PMV-CD11c) and to DEC-205 (PMV-DEC-205). A. After theinjection the draining popliteal lymph nodes were removed for stainingof isolated lymph node cells with a biotinylated anti-CD11c mAb andPE-streptavidin. Flow cytometry dot plots show double staining depictingPE-fluorescence (panels i, iii and v), and correspondingFITC-fluorescence (panels ii, iv and vi) of lymph node cells, asindicated. B. Results of similar experiments in which sections of lymphnode were stained with a biotinylated anti-CD11c mAb andstreptavidin-Rhodamine to identify DCs with the fluorescence images ofcorresponding fields depicting Rhodamine-fluorescence (images i, iii andv), and PMV fluorescein fluorescence (images ii, iv and vi).

FIG. 4 shows that targeting engrafted PMV and SL to DCs stimulates Tcell proliferation. A. Syngeneic C57BL6 splenic T cells were incubatedwith unpulsed DCs, or with DC which had been pulsed with B16-OVA PMVengrafted with L2, CD11c-ScFv, or DEC-205-ScFv (left panel); SL bearingSIINFEKL-6H engrafted with L2, CD11c-ScFv or DEC-205-ScFv (middlepanel); and OVA-containing SL engrafted with L2, CD11c-ScFv orDEC-205-ScFv (right panel). The cells were cultured for 4 days beforeassessing [3H]-thymidine incorporation; results are cpm±SEM. B.Stimulation of CD4⁺ and CD8⁺ T cell proliferation. Syngeneic C57BL6splenic T cells labelled with CFSE were incubated with DCs pulsed withPMV engrafted with DEC-205-ScFv (PMV), SL engrafted with DEC-205-ScFvand SIINFEKL-6H (SIINFEKL-SL), and OVA-containing SL engrafted withDEC-205-ScFv (OVA-SL). The cells were cultured for 4 days and therelative proportion of proliferating CD4⁺ and CD8⁺ T cells, based onCFSE dilution, assessed by flow cytometry.

FIG. 5 comprises the results of the vaccination of mice with PMV and SLand shows stimulation of CTL activity against tumour cells. A. CTLactivity of splenocytes stimulated for 4 days with γ-irradiated B16-OVAcells and derived from mice injected i.v. with PBS alone (PBS), B16-OVAPMV engrafted with L2 peptide (PMV-L2), PMV engrafted with DEC-205-ScFvalone (PMV-DEC-205), or in combination with LPS (PMV-LPS-DEC-205), IFN-γ(PMV-IFN-γ-DEC-205), or GM-CSF (PMV-GM-CSF-DEC-205). B. The CTL activityof splenocytes (25:1 E:T ratio) from mice following immunisation withPMV, SIINFEKL-containing SL, and OVA-containing SL, each engrafted withL2, CD11cScFv or DEC-205-ScFv, as indicated. Results for conditions inwhich LPS, IFN-γ and GM-CSF were incorporated with the engrafted PMV andSL, as indicated, also are shown. Asterisks indicate that CTL activityis significantly higher (n=6. *, P<0.05; **, P<0.01 and **, P<0.001)than mice immunised with a corresponding Ag preparation engrafted withL2 peptide. In panels A and B specific lysis at the indicated E:Tratios, was assessed in a standard ⁵¹Cr release assay. Results areexpressed as the percentage specific lysis±SEM.

FIG. 6 shows that vaccination with modified PMV and SL elicits tumourimmunity. Separate groups of syngeneic C57BL6 mice were immunised (threetimes at weekly intervals) with PMV engrafted with L2, CD11c-ScFv, orDEC-205-ScFv; SL engrafted with SIINFEKL-6H and L2, CD11c-ScFv orDEC-205-ScFv; and OVA-containing SL engrafted with L2, CD11c-ScFv, orDEC-205-ScFv, with each vaccine preparation being injected alone or incombination with LPS or IFN-γ, as indicated. Mice were challenged i.v.with B16-OVA cells, and after 16 days the lungs were removed andexamined for lung metastases. Results show the mean number of tumourfoci for each group of mice±SEM. The dotted line refers to the number oftumour metastases in control mice that were injected with PBS.

FIG. 7 depicts anti-tumour responses in eotaxin knockout mice. A.Syngeneic C57BL6 mice (Eotaxin^(+/+)) or eotaxin knockout mice(Eotaxin^(−/−)) on a C57BL6 background, were immunised with PBS, or withIFN-γ-containing PMV engrafted with either L2 (PMV-L2) or DEC-205-ScFV(PMV-DEC-205), as indicated. Splenocytes were isolated from the mice,and co-cultured with γ-irradiated native B16-OVA cells. Specific lysisat the indicated E:T ratios, was assessed in a standard ⁵¹Cr releaseassay. Results are expressed as the percentage specific lysis±SEM. B.Mice were immunised as above and then challenged i.v. with B16-OVAcells, with the lungs being removed and examined after 16 days fortumour metastases. Results show the mean number of tumour foci for eachgroup of mice±SEM.

FIG. 8 shows that membrane vesicles of BCG mycobacteria engrafted withCD11c-ScFv and DEC-205-ScFv bind to DCs. Ni—(NTA)₃-DTDA was combinedwith PMV derived from BCG mycobacteria and labelled with 6-(fluoresein-5(and -6)-carboxamido)hexanoic acid succinimidyl ester. The PMV were thenengrafted with a control peptide (BCG-Lipo+L2), CD11c-ScFv(BCG-Lipo+CD11c), or DEC-205-ScFv (BCG-Lipo+DEC205), before beingincubated with JAWS-11DC after which cell bound fluorescence wasquantified by flow cytometry. The upper panel shows the results forBCG-Lipo+CD11c while the lower panel comprises the results forBCG-Lipo+DEC205. In each panel, the left-hand trace is for the JAWS-IIcells alone, the middle trace is for the BCG-Lipo+L2 control, while theright-hand trace is for BCG-Lipo+CD11c or BCG-Lipo+DEC205.

FIG. 9 depicts the results of an Elispot analysis of splenic T cellsfrom C57/BL6 mice that had been vaccinated intravenously with engraftedBCG preparations. The engraftments were: L2 peptide as a control (BCGsonicate+L2); CD11c-ScFv (BCG sonicate+CD11c); or DEC-205-ScFv (BCGsonicate+DEC205). Control mice were vaccinated with the PBS used as acarrier for the preparations.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: Ag antigen APC antigenpresenting cell CTL cytotoxic T lymphocyte DC dendritic cell IFN-γinterferon-γ LPS lipopolysaccharide (NTA)₃-DTDA tri(nitrilotriaceticacid) ditetradecylamine OVA ovalbumin PMV plasma membrane vesicle ScFvsingle chain antibody fragment SL stealth liposome

The term “antigen” is used herein to denote any molecule which can betaken up, internalised and processed by DCs, for presentation to theimmune system.

The term “ligand” is used herein to denote any molecule which canspecifically bind in vivo to markers/receptors on the surface of DCs.The term includes whole antibodies, and antibody fragments such as ScFvsand domain antibodies.

The term “immunomodulatory factor” is used herein to denote any “dangersignal”, cytokine or molecule that can modulate the course or outcome ofan immune response.

The term “receptor” is used herein to denote a receptor molecule on thesurface of a DC, and is the entity on the DC surface with which aliposome- or membrane vesicle-engrafted ligand can interact.

The term “tumour” is used herein to denote benign and malignant solidtumours as well as solid and non-solid cancers.

With regard to the first embodiment defined above, theantigen-containing membrane vesicles are typically PMVs but can beformed from any biological membrane or biological structure. The PMVsare advantageously tumour-derived PMVs. The PMVs can also belymphocyte-derived PMVs or leucocyte-derived PMVs. The PMVs can befurthermore membranous preparations of bacteria, protozoa, viruses orfungi. With regard to the antigen-containing liposomes, these includestealth liposomes (SLs) which can be produced from different mixtures oflipids. Such vesicles and liposomes can be prepared as described inreferences 14, 16, 17 and 28, the entire contents of which areincorporated herein by cross-reference.

The Ag of the compositions can be any Ag, or DNA encoding an Ag, againstwhich an immune response is desired. A composition can comprise aplurality of different antigens which may be from the same or adifferent source. That is, a composition comprising tumour antigens mayinclude antigens from different tumours.

The metal chelating groups on the surface of the vesicles and liposomesexist as headgroups of amphiphilic molecules present within thephospholipids and/or lipids comprising the vesicles and liposomes. Theamphiphilic molecule is advantageously nitrilotriacetic acidditetradecylamine (NTA-DTDA) or nitrilotriacetic acidphosphatidylethanolamine (PE-NTA), but compositions can include anymolecule containing any metal binding or chelating moiety that can beincorporated into lipid membranes. Compositions can furthermore comprisemixtures of amphiphilic molecules.

As will be explained in greater detail below, a preferred amphiphilicmolecule is (NTA)₃-DTDA (tri(nitrilotriacetic acid) ditetradecylamine).The related molecule NTA-DTDA, together with other amphiphilic moleculesand vesicles and liposomes containing the same, are described in greaterdetail in International Application No. PCT/AU00/00397 (Publication No.WO 00/64471), the entire content of which is incorporated herein bycross-reference.

The ligand linked to the metal chelating groups on the membrane vesiclesand liposomes can be any metal-affinity tagged molecule that can bindspecifically to any DC surface marker. A preferred metal-affinity tag ishexahistidine. In examples below, hexahistidine-tagged forms of ScFvagainst the DC surface molecules CD11c and DEC-205 (CD205) are used.Other examples include any histidine-tagged ligand such as an antibodyor antibody fragment that can bind to DC surface markers such as DC-SIGN(CD209), CD206 and CD207.

Compositions can include a plurality of ligands for differentmarkers/receptors on DCs. For example, a composition can comprise asligands an ScFv against DEC-205 in combination with an ScFv againstDC-SIGN.

As indicated above, the metal affinity tag of a ligand is typically ahexahistidine moiety covalently linked at a convenient site on theligand. For example, the hexahistidine can be linked to a proteinantigen at the N- or C-terminal thereof. Other metal affinity tagsinclude any moiety or amino acid sequence that can chelate metals andthat can be covalently attached to a convenient site on the ligand.

The immunomodulatory factors of compositions according to the firstembodiment include compounds or molecules that can enhance or modify theresponse of DCs to antigens. Such compounds include “danger signals”(e.g., bacterial lipopolysaccharide), cytokines (e.g., interferon-γ,interleukin-2, interleukin-4, interleukin-10, interleukin-12 andtransforming growth factor-β), as well as chemokine, hormonal and growthfactor-like molecules, or DNA encoding such molecules. A composition caninclude more than one immunomodulatory factor.

Concerning the second embodiment of the invention, suitable processesfor the preparation of membrane vesicles or liposomes with ligandentrapped thereon are described in the international application (No.PCT/AU00/00397) referred to above.

With regard to step (i) of the second embodiment process, the membranevesicles are typically PMVs but can be formed from any biologicalmembrane or biological structure. The liposomes include SLs. The Ag ofthe membrane vesicles and liposomes can be protein, glycoprotein,peptide or polysaccharide, or DNA encoding an antigen, or combinationsthereof, to be delivered to the DCs.

In step (ii) of the second embodiment process, as with the firstembodiment composition, the immunomodulatory factor can be a “dangersignal” (e.g., a bacterial lipopolysaccharide), a cytokine (e.g.,interferon-γ, interleukin-2, interleukin-4, interleukin-10,interleukin-12 and transforming growth factor->), or DNA encoding suchfactors.

The immune response modulation of the method according to the thirdembodiment has application in the prevention or treatment of conditionswhich include transplant rejection, or the effects of autoimmunediseases such as type I diabetes, rheumatoid arthritis, systemic lupuserythematosus and multiple sclerosis. In the case of transplantpatients, this involves the administration of PMVs from donor leukocytesthat are targeted to the DCs of the transplant recipient. Theimmunomodulatory factor in this instance can be, for example, a cytokinesuch as interleukin-10 or transforming growth factor-β. However, theimmunomodulatory factor can be any molecule that has the ability togenerate tolerogenic DCs.

The method of the fourth embodiment can be used in the treatment of anytumour including, but not limited to, melanoma, and cancers of theprostate, bowel, breast and lung. The method can also be used in thetreatment of leukaemia and lymphomas. The method can be used to treattumours in any mammalian animal but is particularly suited for treatingtumours in humans.

The amount of modified Ag-containing membrane vesicles or liposomes tobe delivered to a subject and the administration regime can beestablished by the clinician after assessment of the subject in thelight of the tumour under treatment.

Those of skill in the art will immediately recognise that the methodaccording to the fourth embodiment provides an effective alternative tothe ex vivo manipulation of DCs for use in cancer immunotherapy.

With regard to the fifth embodiment, the method can be used to preventor treat any infection including infections caused by bacteria,mycobacteria, viruses and fungi in order to enhance immunity to theagent responsible for the infection and/or for use in the treatment ofan infection. In a similar fashion to the example given above for theprevention of transplant rejection, all that is required to provide anefficacious method is to prepare PMvs or liposomes that include at leastone antigen from the infectious agent. That antigen can be, for example,envelope proteins of viruses (e.g., HIV, hepatitis B and C) or cell wallcomponents of bacteria (e.g., Mycobacteria), fungi (e.g., Candida) andprotozoa (e.g., malaria).

Administration of compositions to a subject in accordance with the thirdto fifth embodiments of the invention can be by any of the methods knownto those of skill in the art. Compositions are typically administeredintravenously or subcutaneously.

The subject of the methods of the third to fifth embodiments istypically a mammalian subject. The methods are particular suited for usewith a human subject.

Those of skill in the art will appreciate that a medicament according tothe sixth to eighth embodiment of the invention will also include atleast a carrier for the composition. The carrier can be any solutionwith which PMVs and liposomes are compatible. Typical carriers aresaline and buffered saline such as PBS.

Medicaments can include further active agents consistent with theintended use of the medicament. For example, a medicament according tothe seventh embodiment can include other anti-tumour agents while amedicament according to the eighth embodiment can include other agentswith anti-bacterial, anti-protozoan, anti-viral or anti-fungal activityas appropriate for the target infection. Such additional agents will beknown to those of skill in the art.

Prototype Studies

In a prototype study, the inventors have found that the chelator-lipid(NTA)₃-DTDA can be used to anchor His-tagged ScFv onto eithertumour-derived plasma membrane vesicles (PMV) or onto tumourantigen-containing stealth liposomes for the targeting of DCs. Targetingof Ag directly to DCs in this way elicited a strong anti-tumourresponse.

Liposomes have been hailed as having high therapeutic potential, buttheir use has been hampered by a lack of a simple method for attachmentof targeting molecules.¹³ The novel chelator-lipid, (NTA)₃-DTDA (FIG.1A), when incorporated into either SLs or into tumour cell-derived PMV(B16-OVA), enables the stable engraftment of hexa-histidine-tagged ScFvthat target surface molecules on DCs (FIG. 1B and FIG. 1C). PMV and SLsengrafted with ScFv specific for the DC markers CD11c and DEC-205 bindspecifically to DC in vitro and, based on flow cytometry and confocalmicroscopy studies, can target associated Ags directly to DCs in vivo(FIGS. 2 and 3).

Initially, the ability of engrafted PMV and SL to stimulate functionalresponses in assays of DC-initiated Ag presentation was examined. Ourstudies show that ScFv-engrafted PMV and Ag-containing SL aresignificantly more effective than control PMV and SLs at inducing DCs tostimulate T cell proliferation (FIG. 4A). With PMV, proliferation wasstimulated in both CD4⁺ and CD8⁺ T cells. PMV have the potential tostimulate responses mediated by all possible T cell clones reactive toepitopes present in the tumour cell vesicles. Similarly, ScFv-engraftedSL containing the OVA protein may stimulate both OVA-specific CD4⁺ andCD8⁺ T cells; but SL containing SIINFEKL, the immunodominant CTL epitopein OVA, would be expected to generate only CD8⁺ T cell responses.Consistent with this, our data show that DCs targeted by engrafted PMVgenerate approximately equal proportions of CD4⁺ and CD8⁺ T cells,whereas DCs targeted by SL containing SIINFEKL, and to a lesser extentthose containing OVA, generate predominantly CD8⁺ T cells (FIG. 4B).

Evidence suggests that “danger” signals are important in the maturationand migration of DCs after Ag exposure, and can avoid induction oftolerance to the presented Ag.^(9,10,18) Notably, “danger” signals werenot required in the in vivo Ag presentation assays (FIG. 4), presumablysince the DCs are “perturbed” or activated during their isolation. LPSand cytokines like GM-CSF and IFN-γ are known to influence the abilityof DCs to take up Ag and to mature.^(8,19-21) For animal studiestherefore, we incorporated LPS, IFN-γ or GM-CSF, within PMV and SL,thereby providing the means to simultaneously deliver both Ag and adanger signal to DCs.

An examination of the ability of ScFv-engrafted PMV and SL containing Agto induce DCs to initiate CTL responses revealed that, compared tocontrol cells, T cells from animals immunised with ScFv-engrafted PMV orAg bearing SL exhibit an increased ability, following in vitrorestimulation, to lyse B16-OVA target cells in vitro (FIG. 5).Importantly, the results show that in vivo priming for cytolyticactivity is dependent on the presence of “danger” signals, with LPS andIFN-γ stimulating the greatest response (FIG. 5). Both the xenogeneicOVA protein, and a hexahistidine-tagged form of SIINFEKL, could beassociated with SLs for targeting via the engrafted ScFv. Agpresentation and CTL assays thus demonstrate that targetingScFv-engrafted PMV and Ag bearing SLs to DCs in this way can beeffective in stimulating anti-tumour responses, and highlights theimportance of “danger” signals in the induction of immune responses(FIGS. 4 & 5). Moreover, the finding that ScFv-engrafted SL containingSIINFEKL-6H can induce a significant cytotoxic response, demonstratesthat the approach using (NTA)₃-DTDA-containing SLs may be an effectivestrategy for targeting any His-tagged peptide Ag to DCs in vivo.

A finding of paramount importance in this work was our observation thatsyngeneic animals immunised with CD11c-ScFv- and DEC-205-ScFv-engraftedPMV had a significantly lower number of tumour metastases in the lungscompared to controls, after challenge with the B16-OVA melanomaSimilarly, syngeneic animals immunised with ScFv-engrafted SL containingOVA and either LPS or IFN-γ had a lower number of metastases (FIG. 6).The results further show that tumour immunity was completely dependenton the presence of the “danger” signals, LPS and IFN-γ (FIGS. 5 and 6).The immunisation of mice with CD11c-ScFv- and DEC-205-ScFv-engrafted PMVand Ag bearing SL, therefore, target the associated Ag(s) to DCs, whichthen process and present the Ags to T cells inducing Ag-specific T cellactivation, and elicit a strong inhibition in the growth and metastasisof the B16-OVA tumour in vivo. A further significant finding was thefact that, unlike control mice which all developed severe lungmetastases, mice that had been vaccinated with DEC-205-ScFv-engraftedPMV containing IFN-γ after challenge with B16-OVA tumour cellssubsequently did not show any signs of tumour development, indicatingthat the DC targeting vaccine has therapeutic activity.

A particular intriguing aspect of this study is that the apparentgeneration of CTL activity against the B16-OVA melanoma was notassociated with tumour protection. This point is particularly evidentwith the SIINFEKL-SL vaccine that would be expected to generate only aCD8⁺ CTL response against OVA produced by the B16-OVA tumour cells.Despite the vaccine inducing a strong in vitro recall CTL responseagainst B16-OVA tumour cells, no in vivo protection against the tumourwas afforded by the immunisation. It is known that the B16-OVA melanomaline expresses very low levels of MHC class I, and consequently, isresistant to CTL lysis unless high avidity CTLs are used.¹⁴ The factthat splenocytes from mice immunised with DC targeting preparations ofPMV or SL could lyse B16-OVA tumour cells after restimulation withtumour cells in vitro implies that high avidity CTLs can be generatedagainst this tumour cell line. Presumably, such CTLs are either notgenerated, or are not effective in vivo. In fact, previous studiesindicate that CD4⁺ rather than CD8⁺ T cells are effective againstB16-OVA metastases, with CD4⁺ T cells with a cytokine profilecharacteristic of T helper 2 (Th2) cells being particularly effective.¹⁴Furthermore, eotaxin dependent recruitment of eosinophils into thetumours was essential for tumour regression to be observed.¹⁴ To explorea possible role of CD4⁺ T cells-mediated eosinophil recruitment in theanti-tumour effects observed in this study, eotaxin knockout mice wereimmunised with ScFv-engrafted PMV. Our results show that compared tocontrols, eotaxin knockout mice exhibit a markedly reduced ability toinhibit the growth and metastasis of the B16-OVA tumour (FIG. 7A).Eotaxin is a potent eosinophil chemokine and therefore, the findings areconsistent with the recruitment of eosinophils into the tumourconstituting an important component of the anti-tumour response.

The modified PMV and SL system described herein offers a number ofadvantages over current strategies using DCs for tumour immunotherapy.Firstly, the system can deliver Ags directly to DCs in vivo, thuseliminating the need to isolate DCs from patients and to manipulate thecells ex vivo for use in immunotherapies. Secondly, a targeted or activeliposome-mediated delivery of Ag to DC has the potential to deliver moreAg, and/or several different Ags, simultaneously, potentiallystimulating a more effective immune response. The same approach couldpotentially deliver to DCs any Ag or immunostimulatory agent, such as“danger” signals, RNA, DNA, and cytokines, or combinations thereof,which cannot be easily achieved using Ags fused to DC targetingproteins.^(9,10) Thirdly, the approach is versatile and would beconvenient to use clinically since potentially any DC targetingprotein(s) possessing a histidine tag can be engrafted onto the modifiedPMV or SL to deliver specific tumour Ags or other agents to enhancetumour immunity in patients.

Having broadly described the invention and particular applicationthereof in the foregoing prototype studies, specific examples will nowbe given after detailing the materials and methods used therein. It willbe understood by those of skill in the art that these examples are forillustrative purposes only and do not in any way limit the scope of theinvention.

Materials and Methods

Reagents

[³H]-Thymidine and ⁵¹Cr (Na⁵¹CrO₄) were obtained from Amersham(Buckingham-shire, United Kingdom).Palmitoyl-oleoyl-phosphatidyl-choline (POPC), OVA (Grade II, purified byFPLC), LPS (from Escherichia coli serotype 0111:B4), Isopaque, Ficolland P-mercaptoethanol were supplied by Sigma-Aldrich (Castle Hill, NewSouth Wales, Australia). Phosphotidylethanolamine-polyethyleneglycol-2000 (PE-PEG₂₀₀₀) was obtained from Avanti Polar Lipids Inc.(Alabaster).2-(4,4-difluoro-5octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero3-phosphocholine (PC-BODIPY) and 5- (and -6)-carboxyfluoresceindiacetate, succinimidyl ester, mixed isomers (CFSE) were purchased fromMolecular Probes (Eugene, Oreg.). The chelator-lipid NTA)₃-DTDA,consisting of three nitrilotriacetic acid (NTA) head groups covalentlylinked to two ditetradecylamine (DTDA) chains was synthesizedessentially as described,²⁷ but with additional steps to covalentlycouple a NTA group onto each carboxyl group of the NTA-DTDA, to produce(NTA)₃-DTDA. NiSO₄ was used for all additions of Ni²⁺ to buffers.

Monoclonal Antibodies and Proteins

Murine CD56 (clone 42.18, rat IgG2a) mAb was from the 6^(th) Human NKCell Workshop and the murine CD3 mAb (clone 145-2C11, Armenian hamsterIgG) was purchased from PharMingen (San Diego, Calif.). Recombinantmurine IFN-γ and GM-CSF were supplied by PeproTech Inc (Rockey Hill,N.J.). Recombinant ScFv antibodies N418 (anti-CD11c) and NLDC145(anti-DEC-205), each with a hexahistidine (6H) tag at the carboxyterminal and denoted CD11c-ScFv and DEC-205-ScFv, respectively, wereproduced using the baculovirus protein expression system and purified asdescribed.^(16,28) Peptides were synthesised by the BiomolecularResource Facility, John Curtin School of Medical Research (JCSMR), ANU,Can berra. The L2 peptide (GHHPHGHHPH), a sequence of ten amino acidsfound in the plasma protein histidine-rich glycoprotein, was usedroutinely to engraft control PMV and SL since it binds to Ni—(NTA)₃-DTDAwith high avidity and can block its non-specific binding to cells. Thepeptide SIINFEKL-6H, representing the immunodominant CTL epitope of OVAin H-2^(b) mice (OVA residues 257-264), with hexahistidine tag attachedwas used for peptide Ag delivery to DCs.

Mice and Cell Lines

Female or male C57BL6 mice (H-2^(b)) 6-8 weeks of age were supplied bythe Animal Breeding Establishment, (JCSMR, ANU), and C57BL6 eotaxinknockout mice (H-2^(b)) (eotaxin^(−/−)) were a gift from Dr Paul Foster,Division of Biochemistry and Molecular Biology (JCSMR), and were used toobtain lymphoid cells for in vitro assays, and in tumour growth studiesin vivo. The highly metastatic murine B16-OVA melanoma [C57BL6(H-2^(b))], an OVA-secreting tumour cell line was cultured at 37° C. inan atmosphere of 5% CO₂ in RPMI 1640 medium (Gibco-BRL, Invitrogen,Melbourne, Australia) containing 10% fetal calf serum (FCS, TraceBiosciences, Noble Park, Victoria, Australia) and 0.5 mg/mL Geneticin(Invitrogen). Murine Foetal Skin Dendritic Cells (FSDC) [C57BL6-DBA/2JF1 (H-2^(b/d))] were cultured in the same medium but without Geneticin.Murine Long Term Culture Dendritic Cells (LTC-DC) [B10.A(2R)(H-2^(k/b))], isolated and cultured as described,²⁹ were a gift from DrH. O'Neill (School of Biochemistry and Molecular Biology, ANU).

Isolation of Dendritic Cells and T Cells

Murine DC and T cells were isolated from the spleens of C57BL/6 mice.Briefly, splenocytes were isolated by digestion with Collagenase IV(Boerhringer Mannheim), followed by isolation of low density splenocytesby density gradient centrifugation using an Isopaque-Ficoll gradient.DCs were isolated by plastic adherence as described³¹ and then suspendedin complete RPMI 1640 growth medium containing 10% FCS, 5×10⁻⁵ MP-mercaptoethanol, 100 IU/ml penicillin, 100 μg/ml neomycin, and 10 mMHEPES. For isolation of T cells, the spleens were dissociated intosingle cell suspensions, and after removing red cells by hypotoniclysis, the T cells were isolated using a nylon wool column.³²

Plasma Membrane Vesicles and Stealth Liposomes

PMV from cultured cells were prepared by sucrose gradientcentrifugation,³⁰ and modified essentially as outlined.^(16,17)Liposomes used to modify PMV were prepared as follows: ethanolicsolutions of POPC, (NTA)₃-DTDA, LPS and PC-BODIPY (molar ratio94:2:2:2); or POPC, (NTA)₃-DTDA and PC-BODIPY (molar ratio 96:2:2), weremixed, dried under a stream of N₂, then rehydrated in 100 μl PBScontaining 60 μM Ni²⁺. Where indicated, as an alternative to LPS, eitherIFN-γ or GM-CSF (50 ng) was included in the rehydration buffer. Hydratedmixtures were sonicated (three times, 15 sec bursts) using a TOSCO 100Wultrasonic disintegrator (Measuring and Scientific Ltd., London, UK) atmaximum amplitude. Liposomes (100 μl) were mixed with 100 μl of B16-OVAcell-derived PMV (1×10⁸ cell equivalents), before adding 15% PEG₄₀₀ anddiluting 10 times with PBS. The (NTA)₃-DTDA- and cytokine-containing PMVwere purified by size-exclusion chromatography,¹⁷ before engrafting withthe appropriate ScFv.

Stealth Liposomes (SL) were prepared as follows: POPC, (NTA)₃-DTDA,PE-PEG₂₀₀₀, LPS and PC-BODIPY (molar ratio 96:1:1:1:1); or POPC,(NTA)₃-DTDA, PE-PEG₂₀₀₀ and PC-BODIPY (molar ratio 97:1:1:1) dissolvedin ethanol were dried under a stream of N₂, then rehydrated in 100 μlPBS containing 30 μM Ni²⁺ (total lipid 1 mM). For mixtures lacking LPS,IFN-γ or GM-CSF (50 ng) was included in the PBS. Lipid mixtures weresonicated and SL purified (as above). For functional studies thePC-BODIPY was omitted from all lipid mixtures.

Encapsulation of the immunodominant epitope of the OVA protein,SIINFEKL, into SL was attempted but proved difficult since this peptidehas low solubility at the pH used to produce the SL and to engrafthistidine-tagged ScFv (pH 7.4). However, a hexahistidine-tagged form ofthe peptide, SIINFEKL-6H, permitted efficient encapsulation and/orengraftment of the peptide onto (NTA)₃-DTDA-containing SL. Bindingstudies using FACS analysis showed that CD11c-ScFv- orDEC-205-ScFv-engrafted SL containing SIINFEKL-6H could effectivelytarget receptors on DCs in vitro (not shown). Thus, where indicated,SIINFEKL-6H (2 μM) was included to simultaneously engraft with ScFv. Theefficient encapsulation of OVA into SL containing POPC, (NTA)₃-DTDA andPE-PEG₂₀₀₀, was achieved by rehydrating the desiccated lipid mixture inPBS containing 0.1 mg OVA (1 mg/ml), followed by brief sonication. The(NTA)₃-DTDA-containing PMV and SL were engrafted by incubating with theappropriate ScFv (200 μg/ml) for 1 hr at room temperature. The bindingof engrafted PMV and SL to DCs was assessed by flow cytometry aspreviously described.¹⁷

Targeting of DC In Vivo

In order to obtain highly fluorescent PMV for tracking studies PMV werereacted with fluorescein-isothiocyanate (FITC, Molecular Probes),engrafted with L2 or ScFv, and then injected into the hind footpad ofmice. After 16 hrs the draining popliteal lymph node of each animal washarvested and used either for isolation of lymph node cells for twocolour flow cytometric analysis after staining with biotinylated CD11cmAb and streptavidin-phycoerythrin (streptavidin-PE), or for confocalfluorescence imaging. For imaging, lymph nodes were fixed in 10%formalin, then embedded in paraffin, and cut into sections; the sectionswere then adhered onto slides and de-waxed. Slides were blocked byincubation with PBS plus 20% goat serum (PBS-goat serum) for 30 min atroom temperature, before incubating with mAb N418 to CD11c in PBS-goatserum for 1 hr at room temperature. The slides were then washedextensively in water and stained with streptavidin-Rhodamine in PBS-goatserum. After further washing, the slides were analysed for fluoresceinand Rhodamine fluorescence using a Radiance 2000 fluorescence confocalmicroscope (Bio-Rad, Richmond, Calif.). Images were acquired by Kalmanaveraging of 30 successive laser scans, and processed using Bio-RadImage software.

Antigen Presentation Assays

DCs were incubated with modified PMV or SL at 37° C. in complete mediumfor 4 hrs, and then washed to remove unbound PMV or SL, γ-irradiated(5000 rad), and aliquoted in growth medium (2×10⁴ cells/200 μl/well)into a 96-well flat-bottom plate. Syngeneic T cells were added(2×10⁴/well) and the cells co-cultured for 4 days, before assessingproliferation by measuring incorporation of [³H]-thymidine.¹⁴ Theproportion of proliferating CD4⁺ and CD8⁺ T cells in Ag presentationassays was assessed by labelling the T cells with CFSE (5 μM) prior toco-culture with DCs as described.³³ After 4 days co-culture cells werewashed, stained with anti-mouse CD4 (clone L3T4)-Cy-Chrome (10 μg/ml),and anti-mouse CD8 (clone Ly-2)-PE (10 μg/ml), and analysed for CFSE-,Cy-Chrome-, and PE-fluorescence by flow cytometry.

Cytotoxicity Assays

Ag-specific CTL assays were performed similar to those described.³⁴Syngeneic C57BL6 mice were immunized intravenously (i.v.) with PBS(control), or ScFv-engrafted B16-OVA cell-derived PMV or SL bearing Ag(as indicated). At day 14 after immunization, spleens were removed and Tlymphocytes (effector T cells) were isolated as above. The T cells werethen suspended in complete growth medium and aliquoted into 24-wellflat-bottom plates (ICN Biomedicals) at a concentration of 1×10⁵cells/well and co-cultured with 1×10⁵ γ-irradiated (5000 rad) B16-OVAcells. After 5 days of co-culture, the cytolytic activity of the T cellswas assessed in a standard ⁵¹Cr-release assay, as described.¹⁶

Immunisation of Animals and Tumour Challenge In Vivo

Mice were immunized by three i.v. tail vein injections given weekly,with PBS (control), or either ScFv-engrafted B16-OVA cell-derived PMV(2×10⁵ cell equivalents), or SL (˜0.16 μg total lipid) bearingassociated Ag (˜0.2 μg of OVA or ˜0.8 ng of SIINFEKL-6H), each suspendedin a 200 μl volume of PBS. Two weeks after the last injection, the micewere challenged by the i.v. injection of 3×10⁵ B16-OVA cells. At day 16the lungs were removed and the number of tumour foci was countedvisually under a dissection microscope. Alternatively, mice wereimmunised with ScFv-engrafted B16-OVA PMV 3, 6 and 9 days after i.v.injection of 1.5×10⁵ B16-OVA cells.

EXAMPLE 1 Liposomes can be used to Target Tumour Antigens to DC both InVitro and In Vivo

Two types of liposome preparations were used to target tumour Ags to DCs(see FIG. 1 below). The first entailed the use of a crude preparation oftumour cell-derived PMV modified by engraftment of ScFv targeting DC,and the second was a preparation of Ag-containing stealth liposomes alsoengrafted with DC targeting ScFv. Stealth liposomes (SLs) are syntheticlipid structures which have been sterically stabilised by the inclusionof lipids such as PE-PEG₂₀₀₀, and, by virtue of their ability to escapenon-specific elimination by the reticulo-endothelial system, can remainin the blood circulation for days following their intravenousadministration.¹⁴ The use of the chelator lipid NTA-DTDA to modifytumour cells and tumour cell-derived PMV for engraftment of T cellcostimulatory molecules has been described.^(15,16) We have recentlyproduced a novel lipid, (NTA)₃-DTDA (FIG. 1A), which is related toNTA-DTDA, but by achieving a higher local density of NTA headgroups, canpermit a more stable anchoring of histidine-tagged proteins onto bothPMV and onto SLs (not shown). Thus, liposome attachment, via(NTA)₃-DTDA, of histidine-tagged ScFv against DC markers such as CD11cand DEC-205 allows effective targeting of the liposomes to DCs (FIG.1B).

To determine whether liposomes prepared in this manner can be used totarget tumour antigens to DCs, we first explored the ability of thissystem to target Ag to DCs in vitro. In this study we used the highlymetastatic melanoma cell line, B16-OVA, as this line secretes low levelsof OVA which can be used as a surrogate secreted tumour-specific Ag(Ref. 17), enabling OVA-specific immune responses to be assessed. TheB16-OVA tumour line is largely resistant to OVA-specific CTLs unlesshigh avidity CTLs are used.¹⁷ PMV (B16-OVA-derived) could be modified tocontain incorporated (NTA)₃-DTDA by fusion with synthetic liposomescomposed of POPC, (NTA)₃-DTDA, and PC-BODIPY (molar ratio 96:2:2). Also,(NTA)₃-DTDA-containing SLs were produced from an appropriate mixture oflipids: POPC, PE-PEG₂₀₀₀, (NTA)₃-DTDA, and PC-BODIPY (molar ratio96:2:1:1). SLs preparations could be made to contain OVA, or the OVA CTLepitope, SIINFEKL. The (NTA)₃-DTDA-containing PMV and SLs were engraftedwith either a control hexahistidine-containing molecule (L2 peptide) ora hexahistidine-tagged ScFv against either CD11c or DEC-205. Since themodified membranes also contain PC-BODIPY as a fluorescent tracer, theirtargeting to DCs can be assessed by flow cytometry.

Incubation of long term culture DC (LTC-DC) with control-modified PMV(PMV-L2) increased the fluorescence intensity of the cells slightly(˜2-fold above background), but their fluorescence after incubation withPMV engrafted with CD11c-ScFv (PMV-CD11c), or with DEC-205-ScFv(PMV-DEC-205), was 4-8-fold greater than control cells (FIG. 2A). LTC-DCincubated with SL engrafted with CD11c-ScFv (SL-CD11c) and DEC-205-ScFv(SL-DEC-205), also exhibited significant increases in binding (3-6-fold)above control cells (SL-L2) (FIG. 2B). Similarly, the incubation offoetal skin DC (FSDC) that express CD11c, with PMV or SLs engrafted withCD11c-ScFv, resulted in a fluorescence increase substantially above thatof control cells (not shown). The binding specificity of the engraftedPMV and SLs to DCs could be tested using blocking mAbs. Thus,pre-incubation of DCs with an isotype-matched control mAb did notsignificantly reduce binding of either CD11c-ScFv- orDEC-205-ScFv-engrafted PMV or SLs to DC, but their pre-incubation witheither the anti-CD11c mAb N418 or the anti-DEC-205 mAb NLDC145,inhibited binding of the respective ScFv-engrafted SL or PMV by approx.90% (not shown). This demonstrates that the binding is specific for theengrafted ScFv.

To establish that ScFv-engrafted PMV could target DCs in vivo, weinjected mice subcutaneously into the hind footpad withfluorescein-labelled PMV engrafted with ScFv, and then examined cellsisolated from the draining popliteal lymph node for fluoresceinfluorescence by flow cytometry, or sections of the draining lymph nodeby confocal scanning laser microscopy, after PE staining each with aCD11c mAb as a DC marker. The results show that the injection of micewith L2-, CD11c-ScFv or DEC-205-ScFv-engrafted PMV results in a highlevel of CD11c-specific-fluorescence in a relatively small population(2-2.5%) of lymph node cells, thus identifying these as DCs, both byFACS analysis and fluorescence microscopy (FIGS. 3A and B, panels i, iiiand v). Importantly, of the CD11c-positive cells, a greater proportionof fluorescein-labelled cells was seen in the lymph node of miceinjected with ScFv-engrafted PMV (˜1.7%) (FIGS. 3A and B, panels iv andvi), compared to mice injected with L2-engrafted (control) PMV (0.4%)(FIGS. 3A and B, corresponding panels i, and ii). The findings show thatScFv-engrafted PMV can target DCs in vivo. TABLE 1 Liposome and modifiedplasma membrane vesicle preparations Targeting Abbreviation LiposomeType Antigen molecule used Plasma membrane B16 melanoma Control L2PMV-L2 peptide^(c) vesicle (PMV)^(a) antigens + OVA CD11c-ScFv PMV-CD11cDEC-205-ScFv PMV-DEC- 205 Stealth liposome OVA Control L2 OVA-SL- (SL)peptide L2 CD11c-ScFv OVA-SL- CD11c DEC-205-ScFv OVA-SL- DEC-205 Stealthliposome SIINFEKL^(b) Control L2 SIINFEKL- (SL) peptide SL-L2 (OVApeptide) CD11c-ScFv SIINFEKL- SL-CD11c DEC-205-ScFv SIINFEKL-SL- DEC-205^(a)PMV derived from B16-OVA melanoma cell line.^(b)SIINFEKL immunodominant class I MHC epitope with H-2^(b) haplotype.^(c)Control hexahistidine-containing molecule for coupling to liposomes.

EXAMPLE 2 Liposome-Mediated Targeting of Tumour Antigens to DendriticCells Induces Potent Tumour-Specific Immunity Both In Vitro and In Vivo

To determine whether Ag-bearing PMV and SL targeted to DCs can inducefunctional Ag presentation to T cells, we initially examined the abilityof ScFv-engrafted PMV and SL to stimulate T cell proliferation in anAg-presentation assay. Splenic DCs isolated from C57BL/6 mice werepulsed separately with B16-OVA-PMV, SL bearing SIINFEKL-6H, or SLbearing OVA, engrafted with either a control histidine-tagged peptide(L2) or with ScFv against CD11c and DEC-205. After the incubation, thecells were co-cultured with purified syngeneic T cells and then pulsedwith [³H]-thymidine to assess the rate of T cell proliferation. Comparedto control cultures, DCs exposed to PMV or SL (SIINFEKL-6H or OVAbearing) engrafted with CD11c-ScFv induced substantially higher levelsof T cell proliferation. Even greater rates of proliferation were seenwhen the T cells were co-cultured with DC exposed to PMV or SL engraftedwith a DEC-205 ScFv (FIG. 4A). Ag-bearing PMV and SL engrafted withScFv, therefore, can effectively deliver Ag to DCs and stimulate T cellproliferation.

Interestingly, studies using CFSE-labelled T cells revealed that theratio of proliferating CD4⁺ to CD8⁺ T cells was dependent on the Agused. Thus, co-cultures of T cells with DCs which had been pulsed withDEC-205-engrafted PMV consisted of ˜60% CD8⁺ T cells and 40% CD4⁺ Tcells (FIG. 4B). In contrast, co-cultures of T cells and DCs pulsed withDEC-205-engrafted SL bearing the OVA peptide SIINFEKL-6H, contained ˜80%CD8⁺ T cells and ˜20% CD4⁺ T cells, consistent with SIINFEKL being aCD8⁺ T cell epitope. Notably, T cells cultured with DCs pulsed withDEC-205-engrafted SL encapsulating intact OVA contained fewerproliferating CD8⁺ T cells (˜70%) and a significantly higher proportion˜30% of CD4⁺ T cells compared with the SIINFEKL cultures (FIG. 4B),consistent with OVA containing both CD4⁺ and CD8⁺ T cell epitopes. Therelative proportions of proliferating CD4⁺ and CD8⁺ T cells inco-cultures with DCs pulsed with CD11c-ScFv targeted Ags revealed apattern similar to DC pulsed with DEC-205-ScFv targeted Ag (not shown).

Recent studies have demonstrated the importance of danger signals duringAg exposure and DC maturation^(9,10) in determining the type of immuneresponse initiated by DCs. Although studies showed that liposomes cantarget Ag to DCs in vitro and induce T cell responses, previous in vivostudies suggest that for this approach to succeed in vivo, theco-delivery of danger signals to DCs is required. Thus, in order todeliver both Ag and inflammatory stimuli to DCs simultaneously, weproduced Ag-bearing modified PMV and SL that contained incorporated LPS,IFN-γ, or GM-CSF. We found that up to 1% LPS could be included in thelipid mixture, and that PMV and SL could be made to incorporate thecytokines GM-CSF and IFN-γ with high efficiency, without significantlyinterfering with the ability of ScFv engrafted SL to target DCs invitro, as assessed by binding studies using flow cytometry. Moreover,since GM-CSF induces the proliferation of FSDC in serum-free medium, andIFN-γ inhibits their proliferation in complete medium,¹⁷ FSDCproliferation assays were used to monitor cytokine entrapment in the SLwith >85% of the GM-CSF and >75% of IFN-γ being found to be incorporated(not shown).

To determine whether DC-targeted PMV or Ag-containing SL could generateCTL responses in vivo, we immunised C57BL6 mice with preparations thateither lacked or contained danger signals such as LPS, IFN-γ, or GM-CSF.We then isolated splenic T cells, restimulated the cells in vitro withγ-irradiated B16-OVA tumour cells, and assessed their cytolytic activitytowards B16-OVA cells in a standard ⁵¹Cr-release assay. Representativelytic curves are shown in FIG. 5A, for animals that were immunised withvarious PMV preparations engrafted with the DEC-205-ScFv. Little CTLactivity was detected when mice were pre-immunised with PMV engraftedwith the L2 peptide or with DEC-205-ScFv in the absence of a dangersignal (FIG. 5A). Incorporation of either LPS or IFN-γ in theDEC-205-ScFv-engrafted PMV, however, resulted in the induction of highlevels of cytolytic activity, with 50% specific lysis of target cellsstill occurring at a 1:1 effector to target ratio (FIG. 5A). Incontrast, GM-CSF was a much less effective inducer of CTL activity.

For ease of comparison, the cytolytic activity of the various PMV and SLimmunisation conditions are presented at the 25:1 effector to targetratio in FIG. 5B. Maximum CTL activity was observed with splenocytesfrom mice immunised with PMV or SL (SIINFEKL or OVA bearing) containingIFN-γ or LPS as the danger molecule. CD11c-ScFv-engrafted PMV and SLwere somewhat less immunogenic, with GM-CSF being generally a lesseffective danger signal than IFN-γ or LPS but, nevertheless, inducingsignificant CTL activity when associated with PMV, and OVA containingSL. Interestingly, cultures containing T cells from animals injectedwith ScFv-engrafted PMV or SL lacking an associated “danger” signal,gave near background levels of lysis (FIGS. 5A and B).

EXAMPLE 3 Liposome-Based Vaccines that Target DC Induce ProtectiveImmunity Against Tumours

Mice immunised with the various B16-OVA preparations were examined fortheir ability to resist an i.v. challenge of B16-OVA tumour cells, withlung metastases being quantified 16 days following tumour cellinjection. Compared to control mice, a much lower number of metastaseswas observed in mice immunised with PMV or OVA-bearing SL engrafted withScFv and containing either LPS or IFN-γ (FIG. 6). If the PMV orOVA-bearing SL were not engrafted with a ScFv and did not contain LPS orIFN-γ little protection to tumour cell challenge was detected. In starkcontrast, SIINFEKL containing SL were unable to protect mice againsttumour challenge (FIG. 6B), despite some of the vaccine constructsinducing potent CTL activity (FIG. 5). These data are consistent withthe B16-OVA melanoma being resistant to clearance by CD8⁺ CTLs (Ref.14).

To explore the effect of vaccination on pre-existing tumours, weinjected a group of 6 mice with DEC-205-ScFv-engrafted PMV containingIFN-γ at 3 days after challenge with 1.5×10⁵ B16-OVA tumour cells.Interestingly, vaccinated mice subsequently did not show any signs oftumour development, whereas a group of six control animals had to beeuthanised at day 22 due to an increasing tumour burden in the lungswhich contained an average of 250±37 tumour foci each.

The high proportion of proliferating CD4⁺ T cells seen in Agpresentation assays (FIG. 4B), raised the question of whether thesecells, rather than CD8⁺ T cells, play a role in the anti-tumourresponses observed. CD4⁺ T cells recently have been implicated in theclearance of B16-OVA melanoma lung metastases through a mechanisminvolving the eosinophil chemokine eotaxin.¹⁴ The possibility thateosinophils are involved in the anti-tumour response induced bytargeting Ag to DCs was explored in studies in which we immunisedeotaxin knockout mice with PMV-DEC-205-ScFv. The results show thatwhereas the cytolytic activity of T cells from normal and eotaxinknockout mice are essentially identical (FIG. 7A), eotaxin knockout miceimmunised with PMV-DEC-205 exhibit a marked deficiency in their abilityto inhibit tumour growth and metastasis (FIG. 7B).

EXAMPLE 4 Enhancing Immunity to an Infectious Agent by Targeting itsAssociated Antigens to Dendritic Cells

In this example, we demonstrate that the invention can be used to targetantigens of an infectious agent to DCs. BCG is a mycobacteriumcontaining many of the antigens also present in the pathogenMycobacterium tuberculi which is the cause of tuberculosis in humans. Inthe example to be described here, BCG mycobacteria were used instead ofMycobacterium tuberculi. BCG mycobacteria were grown in culture,heat-killed, and labelled [by reacting with a tracer 6-(fluoresein-5(and -6)-carboxamido)hexanoic acid succinimidyl ester] to allowtracking, before modifying to permit targeting to DCs. Thus, theheat-killed BCG was mixed with an appropriate amount of Ni—(NTA)₃-DTDA,and briefly sonicated to permit incorporation of the chelator lipid intothe BCG membrane vesicles containing the BCG antigens. Incorporation ofthe Ni—(NTA)₃-DTDA into the BCG membranes then enabled engraftment ofScFv to either CD11c or DEC-205 to allow specific targeting to the CD11cand DEC-205 markers, respectively, on DCs.

The specific targeting is evident from the graphs comprising FIG. 8. Thefluorescence profiles show that only BCG preparations engrafted with aScFv targeting murine DCs exhibit binding to the murine DC cell lineJAWS-II. There is no binding of the control BCG preparations engraftedwith the non-targeting control protein L2. This indicates that themodified PMVs and liposomes of the invention can be used to targetantigens associated with BCG to DCs in vitro.

Further experiments were conducted to verify that BCG preparationscontaining engrafted DC-targeting ScFv also enhance the immune responseto BCG antigens when used as vaccines in animals. C57/BL6 mice werevaccinated intravenously with the engrafted BCG preparations usingessentially the same vaccination regime as in Example 1 above. After 2-4weeks the mice were sacrificed, their spleens removed for isolation of Tcells and to assay for BCG-specific interferon-γ production. The resultsof an Elispot assay of interferon-γ production were obtained byculturing the T cells isolated from the spleens of the mice in thepresence of heat-killed BCG for a period of three days before assayingthe cultures for interferon-γ-producing cells. The results of theseexperiments are presented in FIG. 9.

It can be seen from FIG. 9 that the spleens from mice vaccinated withBCG preparations that had been engrafted with either of the two ScFvtargeting DCs, show a higher number of interferon-γ-producing T cells(i.e., Elispots) compared to those vaccinated with BCG preparations thathad been engrafted with the control protein L2 (as indicated).Immunomodulatory factors (e.g., interferon-γ, IL-4, IL-10) also can beincluded with the targeted BCG membrane preparations in order to elicitthe most appropriate type of immune response. The results thus show thatas well as targeting antigens to DCs to enhance tumour immunity (asexemplified above), the modified PMVs and liposomes of the invention canalso be used to target antigens from an infectious agent to DCs in vivo,to induce or enhance immunity to the agent.

It will be appreciated by one of skill in the art that many changes canbe made to the methods and compositions exemplified above withoutdeparting from the broad ambit and scope of the invention.

The term “comprise” and variants of the term such as “comprises” or“comprising” are used herein to denote the inclusion of a stated integeror stated integers but not to exclude any other integer or any otherintegers, unless in the context or usage an exclusive interpretation ofthe term is required.

Any reference to publications cited in this specification is not anadmission that the disclosures constitute common general knowledge inAustralia.

REFERENCES

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1. A composition for modulating immunity by the in vivo targeting of anantigen to dendritic cells, the composition comprising: a preparation ofantigen-containing membrane vesicles or antigen-containing liposomeshaving on the surface thereof a plurality of metal chelating groups; anda ligand for a receptor on said dendritic cells, said ligand beinglinked to a said metal chelating group via a metal affinity tag on saidligand; wherein, said antigen-containing vesicles or liposomes includean immunomodulatory factor.
 2. The composition according to claim 1,wherein said antigen-containing membrane vesicles are selected from thegroup consisting of tumour-derived plasma membrane vesicles,lymphocyte-derived plasma membrane vesicles, leucocyte-derived plasmamembrane vesicles, and membranous preparations of bacteria, protozoa,viruses or fungi.
 3. The composition according to claim 1, wherein saidantigen-containing liposomes are stealth liposomes.
 4. The compositionaccording to claim 1, wherein the antigen of said antigen-containingmembrane vesicles or liposomes comprises a plurality of differentantigens.
 5. The composition according to claim 1, wherein said ligandis selected from the group consisting of an antibody, an antibodyfragment and a domain antibody.
 6. The composition according to claim 5,wherein said antibody fragment is a single chain antibody fragment. 7.The composition according to claim 1, wherein said metal-affinity tag onsaid ligand is hexahistidine.
 8. The composition according to claim 1,wherein said immunomodulatory factor is selected from the groupconsisting of a danger signal, a cytokine, a chemokine, an hormonal orgrowth factor-like molecule, and DNA encoding any of the foregoingmolecules.
 9. The composition according to claim 8, wherein said dangersignal is a bacterial lipopolysaccharide.
 10. The composition accordingto claim 8, wherein said cytokine is selected from the group consistingof interferon-γ, interleukin-2, interleukin-4, interleukin-10,interleukin-12 and transforming growth factor-β.
 11. A process forpreparing a composition for modulating an immune response by the in vivotargeting of an antigen to dendritic cells, the process comprising thesteps of: i) preparing antigen-containing membrane vesicles orantigen-containing liposomes; ii) modifying said antigen-containingmembrane vesicles or antigen-containing liposomes by the incorporationof at least one immunomodulatory factor; iii) further modifying saidantigen-containing membrane vesicles or antigen-containing liposomes bythe incorporation of amphiphilic molecules, wherein said amphiphilicmolecules include a chelator group which lies on the surface of saidantigen-containing membrane vesicles or antigen-containing liposomeswhen incorporated therein; and iv) contacting the product of step (iii)with a ligand for a receptor on said dendritic cells, wherein saidligand includes a metal affinity tag for binding to said chelator group.12. The method according to claim 11, wherein said antigen-containingmembrane vesicles prepared in step (i) are selected from the groupconsisting of tumour-derived plasma membrane vesicles,lymphocyte-derived plasma membrane vesicles, leucocyte-derived plasmamembrane vesicles, and membranous preparations of bacteria, protozoa,viruses or fungi.
 13. The method according to claim 11, wherein saidantigen-containing liposomes prepared in step (i) are stealth liposomes.14. The method according to claim 11, wherein said antigen of saidantigen-containing membrane vesicles and antigen-containing liposomes isselected from the group consisting of proteins, glycoproteins, peptides,polysaccharides, and DNA encoding any of the foregoing.
 15. The methodaccording to claim 11, wherein the immunomodulatory factor incorporatedin step (ii) is selected from the group consisting of a danger signal, acytokine, a chemokine, an hormonal or growth factor-like molecule, andDNA encoding any of the foregoing molecules.
 16. The method according toclaim 15, wherein said danger signal is a bacterial lipopolysaccharide.17. The method according to claim 15, wherein said cytokine is selectedfrom the group consisting of interferon-γ, interleukin-2, interleukin-4,interleukin-10, interleukin-12 and transforming growth factor-β.
 18. Themethod according to claim 11, wherein said amphiphilic moleculeincorporated in step (iii) is selected from the group consisting ofnitrilotriacetic acid ditetradecylamine, tri(nitrilotriacetic acid)ditetradecylamine, or nitrilotriacetic acid phosphatidylethanolamine.19. The method according to claim 11, wherein said ligand contacted withthe product of step (iii) is selected from the group consisting of anantibody, an antibody fragment and a domain antibody.
 20. The methodaccording to claim 19, wherein said antibody fragment is a single chainantibody fragment.
 21. The method according to claim 11, wherein saidligand is for a receptor selected from the group consisting of CD11c,DEC-205 (CD205), DC-SIGN (CD209), CD206 and CD207.
 22. The methodaccording to claim 11, wherein said metal-affinity tag on said ligand ishexahistidine.
 23. A method of modulating an immune response in asubject, the method comprising administering to said subject acomposition according to claim
 1. 24. The method according to claim 23,wherein said modulating of an immune response is for the prevention ortreatment of transplant rejection or an autoimmune disease.
 25. Themethod according to claim 24, wherein said autoimmune disease is type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus or multiplesclerosis.
 26. A method of preventing or treating a tumour in a subject,the method comprising administering to the subject a compositionaccording to claim 1, wherein said antigen included in saidantigen-containing membrane vesicles or antigen-containing liposomes isa tumour antigen.
 27. The method according to claim 26, wherein saidtumour is a melanoma, or a cancer of the prostate, bowel, breast orlung.
 28. A method of preventing or treating an infection in a subject,the method comprising administering to the subject a compositionaccording to the first embodiment, wherein said antigen included in saidantigen-containing membrane vesicles or antigen-containing liposomes isan antigen from an agent causing the infection.
 29. The method accordingto claim 28, wherein the causative agent of said infection is abacterium, a mycobacterium, a viruses, or a fungus.
 30. The methodaccording to any one of claims 23 to 29, wherein said subject is a humansubject.